The goal of this program is to develop novel inhibitors of human neutral ceramidase (ASAH2) that are effective as therapeutic agents for Inflammatory Bowel Diseases (IBDs). Sphingolipids are being increasingly recognized as key mediators of inflammation, and are known to mediate the effects of pro-inflammatory cytokines such as tumor necrosis factor-? (TNF?) that are of central importance in IBD. Accumulating evidence demonstrates that sphingosine-1-phosphate (S1P) produced by the combined action of sphingomyelinases, neutral ceramidase and sphingosine kinases within gastrointestinal epithelial and endothelial cells drives the inflammation processes in IBD. Therefore, sphingolipid metabolizing enzymes are potential molecular targets for the development of new drugs for the treatment of IBDs. Because of the pivotal role of ceramidases in regulating inflammation, Apogee Biotechnology Corporation is developing ceramidase inhibitors to treat inflammatory diseases. We have identified the first non-lipid, i.e. drug-like, inhibitors of human ASAH2, which is the dominant ceramidase isozyme expressed in the human gastrointestinal tract. In this Phase 1 SBIR project, we will evaluate the pharmacology and anti-IBD activity of three novel ceramidase inhibitors (representing three chemotypes of ceramidase inhibitors currently being optimized) in two in vivo models of IBD through the following Specific Aims: 1. To synthesize and evaluate the toxicity and pharmacokinetics of novel ceramidase inhibitors. We have identified three chemotypes of drug-like ceramidase inhibitors by high-throughput screening and medicinal chemistry. The lead compound within each of these chemotypes will be synthesized in gram quantities for in vivo testing. The Maximum Tolerated Dose and pharmacokinetics of each of these ceramidse inhibitors will be determined to optimize treatment protocols in the IBD models. 2. To evaluate the therapeutic efficacies of ceramidase inhibitors in the DSS model of ulcerative colitis. Each of the novel ceramidase inhibitors will be tested for efficacy in the dextran sulfate sodium (DSS) model of ulcerative colitis in mice, using a combination of clinical, morphological and biochemical endpoints. 3. To evaluate the therapeutic efficacies of ceramidase inhibitors in the TNBS model of Crohn's Disease. The efficacies of the three novel ceramidase inhibitors will be examined in the TNBS model of Crohn's disease in mice, utilizing similar evaluation endpoints as in Aim 2. This work will provide the first proof-of-principle efficacy studies of ceramidase inhibitors in widely- utilized models of IBD. We have extensive experience with the proposed IBD models and a proven track record for bringing sphingolipid-targeted drugs into clinical trials. We believe that the use of neutral ceramidase inhibitors for te treatment of IBD is an innovative approach that is likely to be rapidly translated to the clinic. PUBLIC HEALTH RELEVANCE: According to the Centers for Disease Control and Prevention, the estimated incidence of IBD in the US is 1.4 million persons, with an overall health care cost of more than $1.7 billion. This chronic condition is without a medical cure and commonly requires a lifetime of care. Over the long term, up to 75% of patients with Crohn's disease and 25% of those with ulcerative colitis will require surgery, making it clear that new therapeutic approaches are needed. Extensive work, including our Preliminary Studies, suggests that inhibition of neutral ceramidase may provide a new therapy for these diseases. The proposed studies will provide the first test of this hypothesis.